Berta, have you tried 100nM and 10min or otherwise been playing with concentrations?

In addition, how do you perform the stimulation, i.e. what medium do you use? Have you done serum starvation for at least 30min?

How do you harvest the cells?

Low doses of insulin rapidly activated Raf-1 by dephosphorylating serine 259 and phosphorylating serine 338 mouse islets.

REFERENCES

Alejandro EU, Kalynyak TB, Taghizadeh F, et al. Acute Insulin Signaling in Pancreatic Beta-Cells Is Mediated by Multiple Raf-1 Dependent Pathways. Endocrinology. 2010;151(2):502-512. doi:10.1210/en.2009-0678.

Boucher J, Kleinridders A, Kahn CR. Insulin Receptor Signaling in Normal and Insulin-Resistant States. Cold Spring Harbor Perspectives in Biology. 2014;6(1):a009191. doi:10.1101/cshperspect.a009191.

Wolfgang,

I think that stimulate mouse islets with 10 or 100nM is not a big difference in terms on activating the signaling may be the time of stimulation is important... so in the next experiment I'll incubate the islets for 10 min

yes, i perform a quite long starvation.

Thank you very much for your answer.

Dear Berta,

We were able to stimulate insulin receptor with 10 uM insulin for 10 min.

You can check my paper:

Selective serotonin reuptake inhibitors (SSRIs) inhibit insulin secretion and action in pancreatic β cells (PMID: 29572328)